2-alkoxy-6-[18F]fluoronicotinoyl substituted lys-c(O)-glu derivatives as efficient probes for imaging of PSMA expressing tissues

ABSTRACT

6-[18F]Fluoro-2-alkoxynicotinoyl substituted Lys-C(O)-Glu derivatives were identified as efficient imaging probes for PSMA expressing tissues in comparison to other known PSMA specific ligands like [18F]DCFPyL, [68Ga]HBED-CC-PSMA, [18F]PSMA-1007 and [Al18F]HBED-CC-PSMA. Unexpectedly, the 6-[18F]fluoro-2-alkoxy and 6-[18F]fluoro-4-alkoxy substituted analogs showed significant differences in accumulation in PSMA expressing prostate tumor cells. Whereas the 2-alkoxy derivative showed cellular uptake values higher than [18F]DCFPyL, the cellular uptake of the corresponding 4-alkoxy substituted derivative was significantly lower. Furthermore, in vivo PET studies with 2-alkoxy-substituted probes demonstrated excellent visualization of PSMA positive ganglia with extremely high target to background ratio. In contrast, the 4-alkoxy substituted derivatives showed less favorable biodistribution with significantly lower uptake in PSMA positive tissues. Especially, the 18F-labeled 2-methoxy derivate ((2S)-2-({[(1S)-1-carboxy-5-[(6-[18F]fluoro-2-methoxypyridin-3-yl)formamido]pentyl]carbamoyl}-amino)pentanedioic acid) demonstrated exceptional clinical efficiency in detecting small PCa lesions, including those which could not be visualized with [68Ga]HBED-CC-PSMA representing currently the gold standard for the diagnosis of recurrent PCa. Furthermore, this probe is easily accessible on a preparative scale in commercially available automated synthesis modules like GE FASTlab and TRACERlab FX N Pro. Consequently, the novel probe is a valuable tool for the visualization of ganglia and reendothelialization as well as for the diagnosis of glioma, neuropathic pain and atherosclerotic plaques.

BACKGROUND OF THE INVENTION

Prostate cancer (PCa) is the most commonly diagnosed cancer and thethird leading cause of cancer-related death among men in Germany with59,620 novel cases and 13,408 deaths in 2013.^([1])2-Deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG), which is an indicator ofglycolytic activity in cancer cells, is generally ineffective in thediagnosis of localized PCa due to the low metabolic glucose activity ofPCa compared with other cancer types.^([2]) Numerous studies revealedthat PCa is associated with changes in fatty acid metabolism. Therefore,[¹¹C]choline, [¹⁸F]fluoromethyl-, or [¹⁸F]fluoroethylcholine, whichtarget upregulated lipid synthesis, have been applied in molecularimaging of PCa.^([3]) However, normal and hyperplastic prostatic tissuesmay also accumulate choline-derived tracers, leading consequently tofalse positive diagnoses.^([4])

PCa is characterized by an elevated level of glutamine metabolism.^([5])Consequently, amino acid PET tracers were utilized for PCa imaging^([6])Especially, anti-1-amino-3-[¹⁸F]fluorocyclobutane-1-carboxylic acid([¹⁸F]FACBC), a conformationally restricted isoleucine analogue,demonstrated promising results in several clinical studies^([7]) and wasapproved by the FDA for the detection of recurrent PCa.^([8]) However, ameta-analysis of data of 251 patients showed a relatively high falsepositive rate for this probe in detecting recurrent PCa, with asensitivity of 87% and a relatively low specificity of 66%.^([9])

Prostate specific antigen (PSMA) expressed by the vast majority ofprostate cancers is a particularly promising target for PCa imagingespecially owing to the correlation of increased PSMA expression withtumor aggressiveness.^([10]) Consequently, PSMA imaging has greatpotential for the improvement of PCa diagnostics and staging. PCa isoften initially diagnosed because of elevated levels of PSA in serum.However, diagnosis should be confirmed by biopsy.^([11]) Frequently, thefirst biopsy fails and needs to be repeated.^([12]) Furthermore, thechoice of treatment, ranging from active surveillance to systemictherapy, should be made on the basis of the grade and stage of atumor.^([12]) Consequently, the ideal procedure for PCa imaging shouldprovide reliable data for disease staging.

At least in Europe, [⁶⁸Ga]Ga-PSMA-HBED-CC (FIG. 1 ) is already widelyused for PCa diagnostics. However, the growing demand for easilyaccessible imaging agents for targeting PSMA stimulated the developmentof several ¹⁸F-labeled PET tracers.^([13]) Among them, [¹⁸F]DCFPyLdeveloped by Chen et al.^([13c]) plays an important role and was studiedin several clinical centers.^([14]) Dietlein et al.^([14a, 15]) reportedthe first comparisons between [¹⁸F]DCFPyL and [⁶⁸Ga]HBED-CC-PSMA inpatients with recurrent PCa. In these studies, [¹⁸F]DCFPyL PET/CTimaging enabled the detection of additional lesions in 21 and 36% of thepatients indicating a higher image quality in comparison to[⁶⁸Ga]Ga-PSMA-HBED-CC PET/CT.

Despite of the fact that [¹⁸F]DCFPyL showed good imaging properties thetracer has some limitations with respect to the detection of very tinylesions and pharmacokinetics. Thus, there is still an unmet need for thedevelopment of even more efficient ¹⁸F-labeled PSMA specific probes withvery high target to background ratio.

It is the objective of the present invention to provide a description ofan innovative PSMA selective PET tracer especially for imaging ofprostate tumor. The objective of the present invention is solved byteaching of independent claims. Further advantageous features, aspectsand details of the invention are evident from the dependent claims, thedescriptions, the figures, and the examples of the presentedapplication.

DESCRIPTION OF THE INVENTION

Subject matter of the present invention is a compound of the generalformula (I):

-   -   wherein R represents C₁-C₁₀ substituted or unsubstituted alkyl,        C₅-C₁₂ unsubstituted or substituted aryl or heteroaryl.    -   In the context of the present invention, an alkyl group, if not        stated otherwise, denotes a linear or branched C₁-C₁₀-alkyl,        preferably a linear or branched chain of one to five carbon        atoms; an alkenyl group, if not stated otherwise, denotes a        linear or branched C₂-C₁₀-alkenyl; and an alkynyl group, if not        stated otherwise, denotes a linear or branched C₂-C₁₀-alkynyl        group, which may be substituted by one or more substituents R′.    -   Preferred alkyl maybe selected from C₁-C₆-alkyl, C₃-C₆-alkenyl        and C₃-C₆-alkynyl.    -   The C₁-C₆-alkyl, C₃-C₆-alkenyl and C₃-C₆-alkynyl residue may be        selected from the group consisting of —CH₃, —C₂H₅, —C₃H₇,        —CH(CH₃)₂, —CH₂—CH═CH₂, —CH₂—C≡CH, —C₄H₉, —CH₂—CH(CH₃)₂,        —CH(CH₃)—C₂H₅, —C(CH₃)₃, —C₅H₁₁, —C₆H₃, —C(R′)₃, —C₂(R′)₅,        —CH₂—C(R′)₃, —C₃(R′)₇, —C₂H₄—C(R′)₃, —C₂H₄—CH═CH₂,        —CH₂—CH═CH—CH₃, —C₂H₄—C≡CH, —CH₂—C≡C—CH₃, —C₂H₄—CH(CH₃)₂,        —CH(CH₃)—C₃H₇, —CH₂—CH(CH₃)—C₂H₅, —CH(CH₃)—CH(CH₃)₂,        —C(CH₃)₂—C₂H₅, —CH₂—C(CH₃)₃, —C₃H₆—CH═CH₂, —C₂H₄—CH═CH—CH₃,        —CH₂—CH═CH—C₂H₅, —CH₂—CH═CH—CH═CH₂, —CH₂—CH═C(CH₃)₂, —C₃H₆—C≡CH,        —C₂H₄—C≡C—CH₃, —CH₂—C≡C—C₂H, —CH₂—C≡C—CH═CH₂, —CH₂—CH═CH—C≡CH,        —CH₂—C≡C—C≡CH, —C₃H₆—CH(CH₃)₂, —C₂H₄—CH(CH₃)—C₂H₅,        —CH(CH₃)—C₄H₉, —CH₂—CH(CH₃)—C₃H₇, —CH(CH₃)—CH₂—CH(CH₃)₂,        —CH(CH₃)—CH(CH₃)—C₂H₅, —CH₂—CH(CH₃)—CH(CH₃)₂, —CH₂—C(CH₃)₂—C₂H₅,        —C(CH₃)₂—C₃H₇, —C(CH₃)₂—CH(CH₃)₂, —C₂H₄—C(CH₃)₃,        —CH(CH₃)—C(CH₃)₃, —C₄H₅—CH═CH₂, —C₃H₆—CH═CH—CH₃,        —CH₂—CH═CH—C₃H₇, —C₂H₄—CH═CH—C₂H₅, —CH₂—C(CH₃)═C(CH₃)₂,        —C₂H₄—CH═C(CH₃)₂, —C₄H₅—C≡CH, —C₃H₆—C≡C—CH₃, —CH₂—C≡C—C₃H₇, and        —C₂H₄—C≡C—C₂H₅;    -   An aryl group denotes an aromatic group having six to twelve        carbon atoms, which may be substituted by one or more        substituents R′, and may be fused to another aromatic ring; the        aryl group is preferably a phenyl group, -o-C6H4-R′, -m-C6H4-R′,        -p-C6H4-R′;    -   A heteroaryl group denotes a 4, 5- or 6-membered heterocyclic        group which contains at least one heteroatom like O, N, S. This        heterocyclic group can be fused to aromatic ring. For example,        this group can be selected from a 3-tetrahydrofuranyl,        3-tetrahydrothienyl, thiazolidinyl, thiadiazolyl, thiazol-2-yl,        thiazol-4-yl, thiazol-5-yl, isothiazol-3-yl, isothiazol-4-yl,        isothiazol-5-yl, oxazol-2-yl, oxazol-4-yl, oxazol-5-yl,        isoxazol-3-yl, isoxazol-4-yl, isoxazol-5-yl,        1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl,        1,2,5-oxadiazol-3-yl, benzoxazol-2-yl, benzoxazol-4-yl,        benzoxazol-5-yl, benzoisoxazol-3-yl, benzoisoxazol-4-yl,        benzoisoxazol-5-yl, 1,2,5-oxadiazol-4-yl, 1,3,4-oxadiazol-2-yl,        1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl,        1,3,4-thiadiazol-2-yl, isothiazol-3-yl, isothiazol-4-yl,        isothiazol-5-yl, benzoisothiazol-3-yl, benzoisothiazol-4-yl,        benzoisothiazol-5-yl, 1,2,5-thiadiazol-3-yl, 1-imidazolyl,        2-imidazolyl, 1,2,5-thiadiazol-4-yl, 4-imidazolyl,        benzoimidazol-4-yl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,        2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl,        3-pyridyl, 4-pyridyl, 2-pyranyl, 3-pyranyl, 4-pyranyl,        2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyrid-2-yl,        pyrid-3-yl, pyrid-4-yl, pyrid-5-yl, pyrid-6-yl, 3-pyridazinyl,        4-pyridazinyl, 2-pyrazinyl, 1-pyrazolyl, 3-pyrazolyl,        4-pyrazolyl, 1,2,3-triazol-4-yl, 1,2,3-triazol-5-yl,        1,2,4-triazol-3-yl, 1,2,4-triazol-5-yl, 1H-tetrazol-2-yl,        1H-tetrazol-3-yl, tetrazolyl, phenazinyl, carbazolyl,        phenoxazinyl, indolizine, 2-indolyl, 3-indolyl, 4-indolyl,        5-indolyl, 6-indolyl, 7-indolyl, 1-isoindolyl, 3-isoindolyl,        4-isoindolyl, 5-isoindolyl, 6-isoindolyl, 7-isoindolyl,        2-indolinyl, 3-indolinyl, 4-indolinyl, 5-indolinyl, 6-indolinyl,        7-indolinyl, benzo[b]furanyl, benzofurazane, benzothiofurazane,        benzotriazol-1-yl, benzotriazol-4-yl, benzotriazol-5-yl,        benzotriazol-6-yl, benzotriazol-7-yl, benzotriazine,        benzo[b]thiophenyl, benzimidazolyl, benzothiazolyl,        quinazolinyl, quinoxazolinyl, cinnoline, quinolinyl,        tetrahydroquinolinyl, isoquinolinyl, or tetrahydroisoquinolinyl,        purinyl, phthalazinyl, pteridinyl, thiatetraazaindenyl,        thiatriazaindenyl, isothiazolopyrazinyl, 6-pyrimidinyl,        2,4-dimethoxy-6-pyrimidinyl, benzimidazol-2-yl,        1H-benzimidazolyl, benzimidazol-4-yl, benz-imidazol-5-yl,        benzimidazol-6-yl, benzimidazol-7-yl, tetrazolyl,        tetrahydro-thieno[3,4-d]imidazol-2-one-yl,        pyrazolo[5,1-c][1,2,4]triazinyl, isothiazolopyrimidinyl,        pyrazolotriazinyl, pyrazolopyrimidinyl, imidazopyridazinyl,        imidazopyrimidinyl, imidazopyridinyl, triazolotriazinyl,        triazolopyridinyl, triazolopyrazinyl, triazolopyrimidinyl, or        triazolopyridazinyl group. This heterocyclic group can be        substituted by one or more substituents R′, wherein R′ is as        defined above;    -   R′ independently represents H, —C₂R″, —CONHR″, —CR″O, —CN,        alkyl, alkoxy, —OH, halogen, haloalkyl or haloalkoxy;    -   A haloalkoxy group denotes an alkoxy group as defined above        substituted by one or more halogen atoms, preferably substituted        by one to five halogen atoms, the haloalkoxy group is preferably        a —OC(R¹⁰)₃, —OCR¹⁰(R^(10′))₂, —OCR¹⁰(R^(10′))R^(10″),        —OC₂(R¹⁰)₅, —OCH₂—C(R¹⁰)₃, —OCH₂—CR¹⁰(R^(10′))₂,        —OCH₂—CR¹⁰(R^(10′))R^(10″), —OC₃(R¹⁰)₇ or —OC₂H₄—C(R¹⁰)₃,        wherein R¹⁰, R^(10′), R^(10″) represent F, Cl, Br or I,        preferably F;    -   In one embodiment R represents C1-C4 substituted or        unsubstituted alkyl. In another embodiment R represents C1-C3        substituted or unsubstituted alkyl. In another embodiment R        represents methyl.    -   In one embodiment R represents

-   -   In one embodiment of the invention the compound of general        formula (I) according to the present invention is for use in        imaging of PSMA-positive organs or tissues or both in a subject.    -   PSMA-positive organs or tissues maybe the following: kidneys,        salivary and lacrimal glands, healing wounds, clear-cell renal        cell carcinoma, glioma, tumor-associated and not associated        neovasculature, lung cancer, glioblastoma multiforme and breast        carcinoma.    -   In one embodiment of the invention the compound of general        formula (I) according to the present invention is for use in        imaging of PSMA-positive organs or tissues or both in a subject        wherein said subject has a pathological condition that is        selected from the group comprising cancer, prostate cancer,        reendothelialization, neuropathic pain and atherosclerosis.    -   In one embodiment of the invention the compound of general        formula (I) according to the present invention is for use in        staging a pathological or physiological condition associated        with one or more PSMA-positive organs or tissues or both of a        subject.    -   In one embodiment of the invention the compound of general        formula (I) according to the present invention is for use in        staging a pathological or physiological condition associated        with one or more PSMA-positive organs or tissues or both of a        subject wherein said subject has a pathological condition that        is selected from the group comprising cancer, prostate cancer,        reendothelialization, neuropathic pain and atherosclerosis.        Neuropathic pain includes peripheral and central neuropathic        pain. Cancer may be selected from the following: clear-cell        renal cell carcinoma, glioma, tumor-associated neovasculature,        lung cancer, glioblastoma multiforme and breast carcinoma.    -   On embodiment of the present invention is a method of making a        compound of general formula (I) according to the present        invention from precursors of a general formula II and        Lys-C(O)-Glu comprising the steps of:

-   -   a) Providing an aqueous solution of [¹⁸F]fluoride;    -   b) Loading of [¹⁸F]fluoride onto a anion exchange resin;    -   c) Washing the anion exchange resin like QMA light (Waters),        ChromaFix PS-HCO₃ (Machery-Nagel), Oasis WAC 3 cc (Waters), QMA        carb (Waters) and Vac QMA 1 cc (Waters) or similar with a polar        aprotic solvent like DMF, DMSO or MeCN, preferably MeCN or C₁-C₆        alcohol preferably MeOH or EtOH or with the mixture of thereof;    -   d) Drying the resin with the flow air or inert gas like He or        Ar;    -   e) Elution of [¹⁸F]fluoride with a solution of a precursor (II)        in a polar aprotic solvent like DMF, DMSO or MeCN, preferably        MeCN or a C₂-C₆ alcohol preferably EtOH or with the mixture of        thereof, preferably MeCN/tBuOH;    -   f) If the elution was carried out using a C₂-C₆ alcohol diluting        the reaction mixture with a polar aprotic solvent like DMF, DMSO        or MeCN, preferably MeCN or aprotic solvent/C₂-C₆ alcohol        mixture, preferably MeCN/tBuOH;    -   g) Heating of the resulting solution at 30-70° C. preferably at        40-50° C. for 1-30 min preferably for 2-7 min which furnishes        the crude [¹⁸F]III;    -   h) Purification of [¹⁸F]III using reversed phase solid phase        extraction (RP SPE) like Chromafix C18 (Machery-Nagel), Sep-Pak        tC18 (Waters) or SepPak HLB (Waters), or similar as follows:        dilution the above mixture with H₂O, loading the resulting        solution on a RP SPE cartridge, washing the cartridge with H₂O,        elution of the purified [¹⁸F]H with C₂-C₆ alcohol, preferably        EtOH;    -   i) Elution of [¹⁸F]I directly to a solution of Lys-C(O)-Glu and        base like CsHCO₃, RbHCO₃, tetraalkylammonium phosphate,        bicarbonate or carbonate preferably tetraalkylammonium        bicarbonate or carbonate most preferably Et₄NHCO₃ in anhydrous        C₂-C₆ alcohol, preferably EtOH;    -   j) Heating the resulting solution at 30-70° C. preferably at        40-50° C. for 1-30 min preferably for 2-7 min;    -   k) Purification of the crude [¹⁸F]I using RP SPE or        alternatively RP HPLC;    -   l) Formulation.

In an alternative embodiment subject matter of the present invention isa method of making a compound of general formula (I) according to any ofclaims 1-4 from a precursor of general formula (IV) comprising the stepsof:

-   -   a) Providing an aqueous solution of [⁸F]fluoride;    -   b) Loading of [¹⁸F]fluoride onto a anion exchange resin;    -   c) Washing the anion exchange resin like QMA light (Waters),        ChromaFix PS-HCO₃ (Machery-Nagel), Oasis WAC 3 cc (Waters), QMA        carb (Waters) and Vac QMA 1 cc (Waters) or similar with a polar        aprotic solvent like DMF, DMSO or MeCN, preferably MeCN or C₁-C₆        alcohol preferably MeOH or EtOH or with the mixture of thereof;    -   d) Drying the resin with the flow air or inert gas like He or        Ar;    -   e) Eluting of [¹⁸F]fluoride with a solution of a precursor (IV)        in a C₂-C₆ alcohol preferably MeOH;    -   f) Evaporation of volatiles;    -   g) Dissolution of the residue in a polar aprotic solvent like        DMF, DMSO, MeCN most preferably MeCN;    -   h) Heating of the resulting solution at 40-130° C. preferably        50° C. for 2-30 min preferably 5 min;    -   i) Addition of 85% H₃PO₄ or 10 M HCl to a solution of the crude        [¹⁸F]V;    -   j) Heating of the resulting mixture at 40-130° C. preferably        50° C. for 2-30 min preferably for 5 min;    -   k) Dilution of the reaction mixture and adjustment of the pH to        2.0-2.5 with an aqueous solution of a base like NaHCO₃, Na₂CO₃,        Et₃N, NaOH, Na₂HPO₄ and Na₃PO₄, preferably Na₃PO₄;    -   l) RP HPLC purification using H₃PO₄ in aqueous EtOH as an        eluent;    -   m) Dilution with isotonic saline, adjustment of the pH with a        base like NaHCO₃, Na₂CO₃, NaOH, Na₂HPO₄ and Na₃PO₄, preferably        NaHCO₃ or Na₂HPO₄;    -   n) Sterile filtration.

In one embodiment of the present invention, the method does not compriseany evaporation steps; and/or wherein the method does not require anydeprotection steps; and/or wherein the method does not require aneutralization step; and/or wherein the method does not require aformulation step.

One embodiment of the present invention is a kit or a cassette systemfor preparing a compound of the general formula (I) said kit or acassette system comprises (i) an anion exchange column; (ii) a reactionvessel; (iii) vials containing aliquots of the appropriate eluents; (iv)a vial containing an aliquot of a precursor compound; (v) reagent vialswherein each reagent vial contains an aliquot of the appropriatereagent; (vi) optionally, one or more SPE columns for purification;(vii) optionally, HPLC column for purification and, (viii) means forcleaning said reaction vessel and said SPE columns.

An anion exchange column maybe selected from the group comprising QMAlight (Waters), ChromaFix PS-HCO₃ (Machery-Nagel), Oasis WAC 3 cc(Waters), QMA carb (Waters), Vac QMA 1 cc (Waters) or similar.

Eluent(s) are preferably a polar aprotic solvent(s) selected from thegroup comprising a polar aprotic solvent like DMF, DMSO or MeCN,preferably MeCN or C₁-C₆ alcohol preferably MeOH or EtOH or with themixture of thereof.

Precursor is a compound according to formula II or formula IV.

The reagent vials contain solvents like MeCN, DMF, DMSO, MeOH, EtOH, H₂Oand saline, solutions of the salts like Et₄NHCO₃ or acids like HCl orH₃PO₄, solutions of precursors like II, IV or Lys-C(O)-Glu.

The reversed phase solid phase extraction (RP SPE) maybe selected fromthe group comprising Chromafix C18 (Machery-Nagel), Sep-Pak tC18(Waters) or SepPak HLB (Waters) or similar.

Means for cleaning the reaction vessels maybe purging with acetone orEtOH and drying under a stream of He, Ar or air.

Subject matter of the present invention is a pharmaceutical compositioncomprising at least one compound of formula (I) together with at leastone pharmaceutically acceptable solvent, ingredient and/or diluent.

Such solvent maybe diluted aqueous EtOH, DMSO, isotonic saline orphosphate buffered saline (PBS).

Such ingredient maybe PEG-400, ascorbinic or gentisic acid.

Such diluent maybe H₂O, isotonic saline or phosphate buffered saline(PBS).

Subject matter of the present invention is a pharmaceutical compositioncomprising at least one compound of formula (I) together with at leastone pharmaceutically acceptable solvent, ingredient and/or diluent foruse in imaging prostate cancer cells or prostate cancerous tissue.

EXAMPLES

Synthesis of Precursors for Radiolabeling

The corresponding onium triflate precursors of radiolabeled activeesters, [¹⁸F]8 and [¹⁸F]9, were prepared by the reaction of2,3,5,6-tetrafluorophenyl 6-chloro-2- or -4-methoxynicotinates (13 and14, respectively) with Me₃N followed by anion metathesis using TMSOTf(FIG. 3 ). 13 and 14 were synthesized from the appropriatechloroanhydrides [obtained by the treatment of 6-chloro-2- or-4-methoxynicotinic acids (17 and 18, respectively) with oxalyl chloridein the presence of DMF traces] and 2,3,5,6-tetrafluorophenol using Et₃Nas a base. 17 and 18 were prepared from 2,6- and 2,4-dichloronicotinicacids, respectively, by the reaction with MeONa, generated in situ fromMeOH and NaH.^([16]) Similarly, the precursor of 2-EtO-6-[¹⁸F]FNic-OTfp(16) was prepared.

Example 01: Preparation of2,3,5,6-tetrafluorophenyl-6-chloro-2-methoxynicotinate(6-C1-2-OMe-Nic-OTfp, 13)

6-Chloro-2-methoxynicotinic acid (17) was prepared according toWO2012/110860 A1 or B. Drennen et al., ChemMedChem 2016, 11, 827-833. Toa suspension of this compound (1.87 g, 9.97 mmol) in anhydrous CH₂Cl₂(10 mL) was added oxalyl chloride (5 mL, 7.4 g, 58.3 mmol) followed byDMF (1 drop, in 5 min an additional drop and in 10 min one more drop).After the vigorous gas evolution was ceased and the solid completelydissolved, the reaction mixture was concentrated using the argon flowand the residue was dried under reduced pressure affording therespective chloroanhydride (2.5 g, 100% crude) which was immediatelyused for the next step. To a solution of this compound in Et₂O (100 mL)was added 2,3,5,6-tetrafluorophenol (1.67 g, 10.06 mmol) followed byEt₃N (1.4 mL, 1.01 g, 10.9 mmol) and the resulting suspension wasstirred for 16 h. Afterwards, the reaction mixture was washed with H₂O(3×20 mL), brine (2×20 mL), dried and concentrated under reducedpressure. The residue was recrystallized from hexane affording 13 (2.4g, 72%) as a colorless solid.

¹H NMR (200 MHz, CDCl₃) δ ppm 4.12 (s, 3H) 7.02-7.12 (m, 2H) 8.38 (d,J=8.0 Hz, 1H)

¹⁹F NMR (188.3 MHz, CDCl₃) δ ppm −138.9 (m), −152.5 (m)

¹³C NMR (50.3 MHz, CDCl₃): δ ppm 55.2, 103.40 (t, J=27.9 Hz), 109.0,116.7, 138.1 (m), 143.5 (m), 144.4, 148.6 (m), 154.6, 159.5, 163.0.

ESI HRMS: calcd for C₃H₅ClF₄NNaO₃ ⁺: 356.97863; found: 356.97959.

Example 02: Preparation of6-methoxy-N,N,N-trimethyl-5-(2,3,5,6-tetrafluorophenoxycarbo-nyl)pyridine-2-aminiumtriflate (6-NMe₃ ⁺-OTf-2-OMe-Nic-OTfp, 7)

13 (1.53 g, 4.56 mmol) was dissolved in 2 M NMe₃ in THF (10 mL; storedover CaH₂) and the resulting solution was stirred for 3 h. A colorlesssolid began to precipitate within first 5 min. After 3 h all volatileswere removed at ≤30° C. using the argon flow and the residue was takenup in anhydrous Et₂O (30 mL) which was removed using the argon flow. Theresidual solid was carefully washed with anhydrous Et₂O and dried underreduced pressure affording the corresponding chloride salt (1.80 g, 100%crude) as a colorless solid which was immediately used for the nextstep.

TMSOTf (2.5 mL, 3.04 g, 13.68 mmol) was added to a suspension of theprepared chloride salt (1.8 g, max. 4.56 mmol) in anhydrous CH₂Cl₂ (10mL) and the mixture was stirred for 30 min. The resulting clear solutionwas concentrated under reduced pressure and the residue was trituratedwith Et₂O and recrystallized from EtOAc affording 6 (1.81 g, 78% overtwo steps) as a colorless solid. The mother liquor was concentratedunder reduced pressure and recrystallized from EtOAc giving the secondcrop of 6 (0.3 g, overall 92%).

¹H NMR (200 MHz, DMSO-d₆) δ ppm 2.88 (s, 9H) 3.38 (s, 3H) 6.70 (tt,J=10.5, 7.3 Hz 1H) 6.90 (d, J=8.2 Hz, 1H) 8.01 (d, J=8.2 Hz, 1H).

¹⁹F NMR (188.3 MHz, DMSO-d₆) δ ppm −155.5 (m), −141.1 (m), −80.05 (m).

¹³C NMR (50.3 MHz, DMSO-d₆) δ ppm 46.2, 46.7, 95.30, 95.8 (t, J=23.4Hz), 98.5, 105.1, 109.1, 115.5, 130.1 (m), 135.6 (m), 138.9, 140.6 (m),149.7 (q, J=45.3 Hz), 154.3.

ESI HRMS: calcd for C₁₆H₁₅O₃N₂F₄: 359.10133; found: 359.10124.

Example 03: Preparation of2,3,5,6-tetrafluorophenyl-6-chloro-4-methoxynicotinate(6-Cl-4-OMe-Nic-OTfp. 14)

6-Chloro-4-methoxynicotinic acid (18) was prepared according to Ehara etal., ACS Med Chem. Len. 2014, S, 787-792. To a suspension of thiscompound (3.87 g, 9.97 mmol) in oxalyl chloride (25 mL, 37 g, 291.5mmol) was added DMF (0.8 mL) followed by anhydrous CH₂Cl₂ (10 mL) andthe reaction mixture was stirred 2 h at 60° C. Afterwards the reactionmixture was concentrated using the argon flow and the residue was driedunder reduced pressure affording the respective chloroanhydride (4.0 g,100% crude) which was immediately used for the next step. To a solutionof this compound in hot EtOAc (100 mL) was added2,3,5,6-tetrafluorophenol (2.92 g, 19.35 mmol; vigorous gas evolutionwas observed). Thereafter, the mixture was cooled to ambienttemperature, Et₃N (2.68 mL, 1.96 g, 19.35 mmol) was added dropwise andthe resulting suspension was stirred for 1 h. Afterwards, the reactionmixture was washed with H₂O (3×20 mL), brine (2×20 mL), dried andconcentrated under reduced pressure. The residue was taken up in CH₂Cl₂(70 mL), the suspension was filtered, the filter cake was washed withCH₂Cl₂ (50 mL). The collected dichloromethane fraction was concentratedunder reduced pressure. The residue was recrystallized from hexaneaffording 14 (2.6 g, 44%) as a colorless solid. The mother liquor wasconcentrated by reduced pressure and the residue was purified by columnchromatography (CH₂Cl₂:hexane=8:2.5) giving the second crop of 14 (0.8g, total 58%).

¹H NMR (200 MHz, CDCl₃) δ ppm 4.04 (s, 3H) 6.89-7.18 (m, 2H) 8.98 (s,1H)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 4.04 (s, 3H) 7.53 (s, 1H) 8.00 (tt,J=10.93, 7.42 Hz, 1H) 8.90 (s, 1H).

¹⁹F NMR (188.3 MHz, CDCl₃) δ ppm −152.4 (m), −138.7 (m).

¹³C NMR (100.56 MHz, DMSO-d₆) δ ppm 57.5, 104.5, 104.8 (t, J=23.6 Hz),109.2, 112.0, 128.3 (m), 138.9 (m), 141.3 (m), 144.3 (m), 146.8 (m),152.9, 157.1, 159.1, 167.1.

ESI HRMS: calcd for C₁₃H₇CF₄NO₃ ⁺: 336.00451; found: 336.00541.

Example 04: Preparation of4-methoxy-N,N,N-trimethyl-5-(2,3,5,6-tetrafluorophenoxycarbo-nyl)pyridine-2-aminiumtriflate (6-NMe₃ ⁺-OTf-4-OMe-Nic-OTfp, 7)

The title compound (1.09 g, 79%; colorless solid) was prepared from 14(1.07 g, 2.71 mmol) using 2 M NMe₃ in THF (10 mL; stored over CaH₂) andTMSOTf (1.44 mL, 1.77 g, 17.96 mmol) as described in Example 02 for 13.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.65 (s, 9H) 4.17 (s, 3H) 7.89 (s, 1H)7.96-8.13 (m, 1H) 9.11 (s, 1H)

¹⁹F NMR (188.3 MHz, DMSO-d₆) δ ppm −153.0 (m), −138.8 (m), −77.7 (m).

¹³C NMR (100.56 MHz, DMSO-d₆) δ ppm 54.7, 58.1, 101.4, 105.0 (t, J=23.6Hz), 114.3, 120.6 (q, J=241.6 Hz), 128.2 (m), 138.7 (m), 141.2 (m),144.4 (m), 146.8 (m), 151.6, 158.7, 161.9, 168.51.

ESI HRMS: calcd for C₁₆H₁₅O₃N₂F₄ ⁺: 359.10133; found: 359.10262.

Example 05: Preparation of 1,5-di-tert-butyl(2S)-2-({[(2S)-1-(tert-butoxy)-6-[(6-fluoro-2-methoxypyridin-3-yl)formamido]-1-oxohexan-2-yl]carbamoyl}amino)pentanedioate(6-F-2-OMe-Nic-Lys(OtBu)-ureido-Glu(OtBu)₂. 20)

A solution of 6 (0.71 g, 1.4 mmol) and H-Lys-OtBu-ureido-Glu(OtBu)₂(0.53 g, 1.09 mmol, prepared according to Mirelli et al., J. Am. Soc.2009, 131, 17090-17092) in anhydrous CH₂Cl₂ (5 mL) was incubated byambient temperature for 72 h. The mixture was concentrated under reducedpressure and the residue was purified by column chromatography (firstMeCN and, thereafter, CH₂Cl₂:MeOH=6:1) affording5-{[(5S)-5-({[(2S)-1,5-bis(tert-butoxy)-1,5-dioxopentan-2-yl]carbamoyl}amino)-6-(tert-butoxy)-6-oxohexyl]carbamoyl}-6-methoxy-N,N,N-trimethyl-pyridine-2-aminiumtriflate (0.65 g, 72%) as a colorless foam, which was directly used forthe next step.

Hexafluorobenzene (82 μL, 132 mg, 0.71 mmol) was added dropwise to asolution of Bu₄NCN (1.15 g, 4.26 mmol) in anhydrous MeCN (4.3 mL), theresulting dark-red solution was stirred to the above triflate (0.59 g,0.71 mmol) and the mixture was stirred for 16 h and taken up with Et₂Oand H₂O (50 mL of each). The ethereal layer was separated and washedwith H₂O (3×20 mL), brine (2×20 mL), dried and concentrated underreduced pressure. The residue was purified by column chromatography(Et₂O) and sonication with pentane to give 20 (0.31 g, 68%) as a viscousyellow oil. R_(f)=0.36 (EtOAc:hexane=1:1).

¹H NMR (300 MHz, CDCl₃) δ ppm 1.10-1.31 (m, 1H) 1.42 (s, 9H) 1.44 (s,9H) 1.44 (s, 9H) 1.46-1.52 (m, 1H) 1.55-1.73 (m, 3H) 1.75-1.93 (m, 2H)1.97-2.17 (m, 1H) 2.18-2.45 (m, 2H) 3.43 (d, J=6.3, 19.6 Hz, 2H) 4.08(s, 3H) 4.20-4.45 (m, 2H), 4.79-5.78 (br, 2H) 6.62 (dd, J=8.2, 3.1 Hz,1H) 7.76 (t, J=5.50 Hz, 1H) 8.63 (t, J=8.2 Hz, 1H).

⁹F NMR (282 MHz, CDCl₃) δ ppm −65.45 (dd, J=8.2, 2.7 Hz).

¹³C NMR (75.5 MHz, CDCl₃): δ ppm 22.5, 27.95, 27.97, 28.03, 28.5, 29.1,31.5, 32.3, 39.3, 52.9, 53.4, 54.9, 80.5, 81.6, 81.9, 101.7 (d, J=35.5Hz), 113.2 (d, J=5.3 Hz), 146.9 (t, J=9.1 Hz), 156.9, 159.9 (d, J=14.3Hz), 162.8 (d, J=246.9 Hz), 163.0, 172.0, 172.3, 172.4.

Example 06: Preparation of(2S)-2-({[(1S)-1-carboxy-5-[(6-fluoro-2-methoxypyridin-3-yl)formamido]pentyl]carbamoyl}amino)pentanedioicacid (1)

A solution of 20 (0.31 g, 0.66 mmol) in TFA/TIS/H₂O=95/2.5/2.5 (10 mL)was incubated for 90 min at ambient temperature. Afterwards, allvolatiles were removed under reduced pressure and the residue was takenup in TFA (10 mL), the resulting solution was incubated at ambienttemperature for 3 h and concentrated under reduced pressure. The residuewas sonicated with Et₂O and recrystallized from MeOH/Et₂O affording 1(80 mg, 36%) as a colorless solid. The mother liquor was concentratedunder reduced pressure and the residue was recrystallized from MeOH/Et₂Oto give the second crop of the title compound (45 mg, overall 56%).

¹H NMR (300 MHz, CD₃OD) δ ppm 1.43-1.57 (m, 2H) 1.58-1.80 (m, 3H)1.81-1.98 (m, 2H) 2.07-2.22 (m, 1H) 2.32-2.49 (m, 2H) 3.41 (t, J=6.87Hz, 2H) 4.06 (s, 3H) 4.22-4.37 (m, 2H) 6.70 (dd, J=8.15, 2.89 Hz, 1H)8.39 (t, J=8.15 Hz, 1H).

¹⁹F NMR (282 MHz, CD₃OD) δ ppm −67.67 (dd, J=7.80, 3.04 Hz).

¹³C NMR (75.5 MHz, CD₃OD): δ (ppm) 24.2, 29.1, 30.2, 31.2, 33.3, 40.8,53.7, 54.2, 55.5, 102.3 (d, J=36.2 Hz), 115.2 (d, J=5.3 Hz), 147.1 (d,J=9.1 Hz), 160.3, 161.9 (d, J=14.3 Hz), 162.94 165.71 (d, J=209.1 Hz),166.2, 175.9, 176.5, 176.6.

ESI HRMS: calcd for C₁₉H₂₅O₉N₄FK⁺: 511.12372; found: 511.12366; calcdfor C₁₉H₂₅O₉N₄FNa⁺: 495.14978; found: 495.14959; calcd for C₁₉H₂₆O₉N₄F⁺:473.16784; found: 473.16756.

Radiosynthesis of [¹⁸F]1 and [¹⁸F]2

The novel PET tracers, [¹⁸F]1 and [¹⁸F]2, and [¹⁸F]DCFPyL ([¹⁸F]4) wereprepared by the acylation of Lys-CO-Glu urea 10 with the appropriate¹⁸F-labeled active ester, [¹⁸F]8 and [¹⁸F]9, in EtOH using Et₄NHCO₃ as abase (FIG. 2 ).[¹⁷] The corresponding radiofluorinated active esterswere prepared by the elution of the [¹⁸F]fluoride loaded on a anionexchange resin with a solution of the radiolabeling precursor in asuitable solvent (EtOH, EtOH/MeCN/tBuOH, MeCN/tBuOH or MeCN) followed byheating of the resulting solution to 40° C. for 2-5 min (if pure EtOHwas used as an eluent, it was preliminary diluted with MeCN/tBuOHmixture). The crude radiolabeled active esters were purified by solidphase extraction Alternatively, [¹⁸F]1 and [¹⁸F]2 could be preparedusing the one-pot two-step procedure similar to that proposed for thepreparation of [¹⁸F]DCFPyL by Bouvet et al.^([18]) and Ravert etal.^([19]) [⁶⁸Ga]Ga-PSMA-HBED-CC was synthesized according to Eder etal.,^([20]) [¹⁸F]AlF-PSMA-HBED-CC and PSMA-1007 were produced accordingto Boschi et al.^([21]) and Cardinale et al.^([22]), respectively.

Example 07: Manual Synthesis of [¹⁸F]1

Aqueous [¹⁸F]fluoride (0.05-50 GBq) was loaded onto a Sep-Pak AccellPlus QMA carbonate plus light cartridge (Waters GmbH, Eschborn, Germany)preconditioned with 1 mL EtOH followed by 10 mL H₂O. The resin waswashed with anhydrous EtOH (3 mL) and [¹⁸F]fluoride was eluted into thereaction vessel with a solution 6 (10 mg, 21 μmol), in anhydrous EtOH(200 μL). The resin was then washed with anhydrous MeCN/tBuOH 1:4 (2 mL)into the reaction vessel too. The mixture was allowed to stir at 45° C.for 15-20 min. After that, the crude mixture was diluted with water (10mL) and the solution was loaded onto a polymer RP or C-18 cartridge. Thecartridge was washed with water (10 mL) and [¹⁸F]8 was eluted with EtOH(500 μL). Alternatively, the anion exchange resin was washed withanhydrous MeCN (3 mL) and [¹⁸F]fluoride was eluted into the reactionvessel with a solution 6 (12 mg, 25 μmol), in anhydrous MeCN/tBuOH 1:4(0.6 mL). The resin was then flushed with anhydrous MeCN/tBuOH 1:4 (1mL) into the reaction vessel, too. The mixture was allowed to stir at40° C. for 1-3 min, diluted with water (10 mL) and the solution wasloaded onto a polymer RP or C-18 cartridge. The cartridge was washedwith water (10 mL) and [¹⁸F]8 was eluted with EtOH (500 μL) directly toa solution of Lys-C(O)-Glu (2.5 mg, 7.8 μmol) in 0.19 M Et₄NHCO₃ inanhydrous EtOH (160 μL) and the reaction mixture was allowed to stir for3-5 min at 45° C. The mixture was quenched with 0.1% TFA (20 mL) andloaded onto a preconditioned Sep-Pak C18 plus long cartridge. Thecartridge was washed with water (10 mL) and after that plugged to a SepPak HLB short cartridge. [¹⁸F]1 was transferred from the C18 onto theHLB resin by 1.7% H₃PO₄ in 6% EtOH (60 mL). HLB cartridge was washedwith water (10 mL) and the product [¹⁸F]PSMA-7 eluted with 50% EtOH inisotonic saline (2 mL). (FIG. 7 )

Example 08: Automated Production of [¹⁸F]1 on FXNPro Module (GE)Starting from [¹⁸F]Fluoride without HPLC Purification

Aqueous [¹⁸F]fluoride (0.05-50 GBq) was transferred from the cyclotrontarget into a trapping vial and thereafter loaded onto a anion-exchangeresin cartridge (Sep-Pak QMA carbonate light 46 mg, preconditioned with1 mL water) from the male side of the cartridge. [¹⁸O]H₂O was collectedin a separate vial. The cartridge was subsequently washed with MeCN (4mL) from vial V1 from the female side of the cartridge. Washings werediscarded. Thereafter, [¹⁸F]fluoride was slowly eluted from the resinwith a solution of 6 (10 mg, 20 μmol) in tBuOH:MeCN (4:1) (1 mL) fromthe vial V2 into reactor R1 using a stream of He. Afterwards, MeCN (2mL) from vessel V3 was passed through the cartridge into reactor R1.Reactor R1 was filled with He, sealed and the reaction mixture washeated at 45° C. for 3 min. After cooling to ambient temperature thereaction mixture was diluted with H₂O (15 mL) from vessel V5 and loadedonto a polymer RP cartridge (Strata X, preconditioned with 1 mL EtOHfollowed by 5 mL H₂O). The cartridge was washed with H₂O (10 mL) fromvial V4D and dried using a flow of helium for 5 min.

[¹⁸F]8 was eluted with a freshly prepared solution of Lys-C(O)-Glu (4.6mg, 15.2 μmol) and Et₄NHCO₃ (11.6 mg, 60.6 μmol) in EtOH (1 mL) fromvial VX4 into reactor R2. The reaction mixture was heated at 40° C. for3 min. After cooling to ambient temperature the reaction mixture wasdiluted with water (1 mL) from vial V7 and transferred to vessel CV3containing 0.1% TFA (20 mL). The acidic solution was loaded onto a tC18cartridge (Sep-Pak tC18 Plus Long Cartridge, 900 mg, preconditioned with10 mL EtOH followed by 30 mL ‘H₂O). The cartridge was subsequentlywashed with water (10 mL) from vial V35 and [¹⁸F]1 was eluted with 1.7%H₃PO₄ in 12% EtOH (60 mL) onto a HLB catridge (Oasis HLB Plus ShortCartridge 225 mg, preconditioned with 10 mL EtOH followed by 30 mL H₂O)from vessel V9. The HLB cartridge was washed with 10 mL water fromvessel V43 and the purified [¹⁸F]1 was eluted with 50% EtOH in isotonicsaline (2 mL). The resulting solution was diluted with isotonic saline(9 mL) and sterile filtered. Quality control: eluent: 1.7% H₃PO₄ 10%EtOH for 5 min, then 50% EtOH for 2 min. Flow rate: 3 mL/min. Column:Chromolith® SpeedROD RP-18e column (Merck, Darmstadt Germany), 50×4.6mm. Retention times: [¹⁸F]1=3 min; [¹⁸F]6=5.7 min.

Example 09: Automated Synthesis of [¹⁸F]1 on GE FASTlab

Assembling of the Cassette

The cassette for the production of [¹⁸F]1 (FIG. 8 ) was assembled usingoriginal components available from GE. First, the spike at B wasconnected by short flexible silicone tubing with the female side of aQMA carbonate light cartridge installed at C. At position A a shortflexible silicone tubing was mounted for connection with the [¹⁸O]H₂Ocollection bottle. At positions D, O and U long flexible siliconetubings were installed to connect the cassette with large solventstorage bottles. A polymer reactor was connected using short siliconetubing to positions F, G and by long tubing to position V (the middleriser of the reactor was connected to valve G). At position H a smallCis cartridge was installed (Chromafix C18 ec, 250 mg) with the femaleside connected by long silicone tubing to R. At I, a 10 mL disposablevial with isotonic saline was installed using short silicone tubing. Inslots J, K, L, and N small reagent vials were installed. Two types ofreagent vials were used: 3 mL glass vial with 11 mm neck (260 μL deadvolume), and a 5 mL glass vial with 13 mm neck (250 μL dead volume). Thevials were closed with a rubber stoppers and crimped with aluminum caps.

During filling of the vial with the appropriate reagents, the deadvolume had to be taken into account. For example, the 3 mL vialcontaining [¹⁸F]6 precursor had to be filled with 23 mg precursorinstead of the actually needed 10 mg.

However, filling of the vial with 460 μL EtOH would be equal to asolution of 10 mg precursor/200 μL (as it is required for the synthesis)plus a non-recoverable fraction of 260 μL. Vial J was a 3 mL type,containing 460 μL EtOH and 23 mg precursor 6. Vial K was a 5 mL type,containing 2.25 mL tBuOH/MeCN 4:1. Vial L was a 5 mL type and containeda solution of Lys-C(O)-Glu (2.5 mg, 7.8 μmol) and Et₄NHCO₃ (5.7 mg, 30μmol) in anhydrous EtOH (750 μL). Vial N was a 5 mL type and contained4.5 mL of 0.5% aqueous TFA. Slot M was equipped with a special spikeused for the connection to a sterilized water bottle. At position P aSep Pak C18 plus long cartridge was mounted with the female sideconnected by short silicone tubing to Q. At position T an Oasis HLB PlusShort Cartridge was mounted with the female side connected by a shortsilicone tubing to position S.

Radiosynthesis (cf FIG. 9 )

The synthesizer was reseted and the self-check was performed by default.After passing the preliminary tests, the cassette was mounted and aprogrammed cassette self-test was performed to confirm leak-tightness ofthe cassette components. After passing this test, the tubing at D(EtOH), I (saline) and O (phosphoric acid) were connected. A 250 mLsterilized water bottle was connected via the spike at position M. LinesA and U were connected to the [¹⁸O]H₂O collection vessel and productvial, respectively. Afterwards, a programmed procedure was started toactivate the SPE cartridges: the QMA carbonate light cartridge at C waspreconditioned with H₂O (1 mL). The Chromafix C18 RP cartridge at H waspreconditioned with EtOH (1 mL) followed by H₂O (3 mL). The Sep-Pak C18Plus Long Cartridge at P and Oasis HLB Plus Short Cartridge at T werepreconditioned with EtOH (3 mL) followed by H₂O (20 mL). All reagentvials except the storage bottles at D, I, O were pressurized with helium(+1000 mbar). After these preliminary steps, the cassette was ready tostart the synthesis.

Irradiated ¹⁸O-water (1.5 mL) was transferred from the cyclotron targetto the receiver vial at position E and loaded onto the QMA carbonatelight cartridge at C. The cartridge was subsequently washed with EtOH(2×1 mL from storage vessel D) by activation of S1. Thereafter,[¹⁸F]fluoride was eluted stepwise into the reactor from the resin with asolution of 6 in EtOH (200 μL) stored in vial J. MeCN/tBuOH 1:4 (2 mL)from vial K was passed through the QMA cartridge into the reactor.Afterwards, lines were flushed with helium into the reactor to recoverany residual activity. Thereafter, the reactor was sealed and heated at50° C. for 15 min.

The reactor was charged by a constant low flow of helium to ensurepressure equalization, and the reaction mixture was quenched with H₂O (2mL) from reservoir M by activation of syringe 2 (S2). Syringe 2 wasfilled with H₂O (4 mL), an aliquot of crude [¹⁸F]8 (500 μL) and air (500μL) to ensure proper mixing. The solution was loaded onto the C18 resinat position H. This stepwise dilution procedure was performed at leastfour times until full recovery of the reaction mixture in the reactorhas been achieved. The C18 cartridge was washed with H₂O (10 mL) anddried with a stream of helium (15 s). Afterwards, the reactor, themanifold, tubing H→R and yringe 1 were thoroughly cleaned with EtOH.Consequently, purified [¹⁸F]8 was eluted into the reactor from the C18resin with a solution of Lys-C(O)-Glu and to Et₄NHCO₃ in EtOH from thestorage vessel at position L. Then, the reactor was sealed and heated at40° C. for 3 min and supplied with a constant low flow of helium toensure pressure equalization. Subsequently, the reaction mixture wasquenched with 0.5% TFA (4 mL). The acidic solution of crude [¹⁸F]1 wasloaded onto the Sep-Pak tC18 Plus Long cartridge at position P. Thecartridge was washed with H₂O (2×5 mL) and dried by applying a high flowof nitrogen (15 s). The C18 cartridge was switched in line with theSepPak HLB plus short at position T and 1.7% H₃PO₄ in 12% EtOH (60 mL)from the storage vessel connected by tubing at O was passed through bothcartridges, and [¹⁸F]1 was trapped onto the HLB resin. The HLB cartridgewas washed with H₂O (10 mL) and purified [¹⁸F]1 was eluted with ethanol(500 μL) from reservoir D into storage syringe 3 (S3). An ethanolicsolution of [¹⁸F]1 in syringe 3 was diluted with isotonic saline (10 mL)from vessel I. The resulting radiotracer solution was dispensed uponrequest into a vial at position U.

Example 10 Biological Evaluation of [¹⁸F]1 and [¹⁸F]2 in Comparison toKnown PSMA Specific Tracers

Within our ongoing program on the development of the novel PSMA-specificPET ligands we prepared and evaluated 6-[¹⁸F]fluoro-2- and4-methoxynicotinoyl substituted probes (2- and 4-MeO-[¹⁸F]PSMA, [¹⁸F]1and [¹⁸F]2, respectively) (FIG. 1 ). The biological properties of thenovel compounds were compared with those of the known PSMA-specific PETtracers such as [⁶⁸Ga]Ga-PSMA-HBED-CC, [¹⁸F]DCFPyL,[¹⁸F]AlF-PSMA-HBED-CC^([21]) and [¹⁸F]PSMA-1007.^([23])

Cellular Uptake Experiments

The cellular uptake of the novel PET probes in PSMA positive LNCaP C4-2and PSMA negative PC3 prostate tumor cell line in the presence andabsence of 2-(phosphonomethyl)pentanedioic acid (2-PMPA; 20), knownnanomolar PSMA inhibitor,^([24]) was measured and compared with that of[¹⁸F]DCFPyL carried out in parallel (FIG. 4A). 2-MeO-[¹⁸F]PSMAdemonstrated a much higher uptake in PSMA⁺ LNCaP C4-2 cells than[¹⁸F]DCFPyL after 2 h incubation (2.03±0.03 vs. 1.55±0.05% ID/10⁵cells). The difference in the accumulation of the both tracers in thesame cells after 4 h incubation was less pronounced (3.31±0.01 and3.1±0.03% ID/10⁵ cells for 2-MeO-[¹⁸F]PSMA and [¹⁸F]DCFPyL,respectively). The PSMA specificity of the tracer uptake in LNCaP C4-2was confirmed by complete inhibition with 2-PMPA (≤0.1% ID/10⁵ cells).The enrichment of both tracers in PSMA⁻ PC3 cells was negligible (≤0.1%ID/10⁵ cells). In contrast, the cellular uptake of 4-MeO-[¹⁸F]PSMA inLNCaP C4-2 cells was significantly lower than that of [¹⁸F]DCFPyL(0.79±0.04 vs. 1.53±0.03% ID/10⁵ cells (after 2 h incubation) and0.90±0.04 vs. 3.16±0.01% ID/10⁵ cells (after 4 h incubation) (FIG. 4B).As in the case of 2-MeO-[¹⁸F]PSMA the intracellular accumulation of4-MeO-[¹⁸F]PSMA in LNCaP C4-2 cells was completely blocked by 2-PMPA andwas very low in PSMA negative PC3 cells.

Example 11: Cellular Uptake of [¹⁸F]1 and [¹⁸F]2 in PSMA⁻ PC-3 Cells andPSMA⁺ LNCaP C4-2 Cells

Cell culture: PC3 and LNCaP C4-2 prostate tumor cells were generousgifts of G. Winter (Ulm, Germany).

PSMA⁻ PC-3 cells were cultured in RPMI-1640 medium supplemented with FBS(10%) and penicillin/streptomycin (1%). PSMA* LNCaP C4-2 cells werecultured in a mixture of DMEM: Ham's F-12K (Kaighn's) mediums (4:1)supplemented with FBS (5%), NaHCO₃(3 g/L), insulin (5 μg/mL),triiodothyronine (13.6 μg/mL), transferrin (5 μg/mL), biotin (0.25μg/ml) and adenine (25 μg/mL). Both cell lines were grown in 75 mLflasks containing 10 mL of the culture medium in a humidified atmosphereof 5% CO2/95% air at 37° C. for 4-5 days until they reached 80-90%confluency. Cells were seeded into 12-well plates (1×10⁵ cells/wellcontaining 1 mL medium) 24 h before the beginning of the cellular uptakeexperiments.

The corresponding PSMA specific PET probe was added to the cells(100-150 kBq/well) and the cells were incubated at 37° C. for 1 and 2 h.2-(Phosphonomethyl)pentanedioic acid (2-PMPA; 100 μM/well) was used forblocking studies. Thereafter, the cells were washed two times withmedium (1 mL), trypsinized, harvested and the accumulated radioactivitywas measured in a gamma counter (Wizard 1470, PerkinElmer,Massachusetts, USA). The cellular uptake of the novel PSMA-selectivecandidates and [¹⁸F]DCFPyL obtained in experiments performed in parallelwere compared. Each cellular uptake experiment was carried out intriplicate.

PET Study of PSMA-Specific Tracers in Healthy Rats

Ganglia represent an ideal tissue for the evaluation of PSMA bindingimaging probes. Having a size of 1-2 mm these structures aresufficiently small to imitate metastases in a very early stage.Ganglionic PSMA is expressed by satellite glial cells, which envelop theneuronal cell bodies of the trigeminal ganglion,^([25]) spinal dorsalroot ganglia and ganglia of the autonomic nervous system.^([26])Electron microscopic studies showed that PSMA protein is mainlylocalized in the cell membrane of satellite cells.^([27]) Rat PSMAcomprises 752 amino acids (vs. 750 in humans), and has about 91%homology to the human PSMA. Importantly, all amino acid residues of theactive site are essentially the same as those in the human homologuewith the only exception of Gly⁵⁴⁸ in the human and Ser⁵⁴⁸ in the ratprotein, respectively^([28]) Furthermore, rat and human PSMA showcomparable kinetic parameters for hydrolysis ofN-acetylaspartylglutamate (NAAG) and similar inhibition profiles.

Notably, in contrast to tumor xenografts which rapidly change over timeconcerning volume, degree of vascularization and necrosis, ganglia asnatural PSMA-expressing tissues remain constant over a long time. Thisallowed evaluation of all tested PSMA ligands in the same animal undersimilar conditions. (FIG. 5 )

TABLE 1 Comparison of the different PSMA-specific tracers SCG SCGsignal-to- Liver Bone % ID noise ratio % ID % ID [¹⁸F]DCFPyL 20.2 ± 5.8 6.7 ± 2.6  65.3 ± 21.7 12.4 ± 4.3  (n = 6) [¹⁸F]DCFPyL + 4.6 ± 1.8  3.7± 2.1 47.0 ± 7.7 6.1 ± 1.9 2PMPA (n = 3) [¹⁸F]AlF-PSMA- 36.8 ± 9.5  4.5± 1.4  15.5 ± 4.2† 122.8 ± 50.2§ HBED-CC (n = 3) [⁶⁸Ga]PSMA- 41.0 ±3.4*  4.5 ± 0.1  47.0 ± 10.3 18.5 ± 0.6  HBED-CC (n = 3) 2-MeO- 31.3 ±10.5  8.2 ± 1.7 119.3 ± 8.3‡ 9.9 ± 1.9 [¹⁸F]PSMA (n = 3) 4-MeO- 14.4 ±2.6  4.6 ± 1.3  29.0 ± 7.2† 19.2 ± 2.1  [¹⁸F]PSMA (n = 3) [¹⁸F]PSMA-100794.8 ± 19.6* 6.2 ± 1.9 50.7 ± 4.3 33.2 ± 9.5  (n = 3)

Conditions: Uptake of different PSMA-specific tracers in healthy ratsmeasured by PET. Conditions: PET scanner (Focus 220, Siemens); 57-71 MBqtracer was injected. PET scans started 60 min after injection andcontinued for 60 min. % ID was determined by dividing each image by theinjected dose and multiplying it by body weight. Elliptical volumes ofinterest (VOIs) were drawn to extract mean % ID values for the rattrigeminal and superior cervical ganglia (9 mm³). Background activityfor calculation of signal-to-noise ratio was measured dorsal from thecervical vertebral column with a 390 mm³ VOI.

[¹⁸F]DCFPyL accumulated in peripheral ganglia with the strongest uptakein the ganglion of the trigeminal nerve (37.9±9.9% ID; n=6) measured60-120 min after injection. In the overlay with the CT image, eightfocal accumulations of radioactivity were detected in theinterventricular forming between the cervical vertebrae, an anatomicallocalization assigned to the spinal dorsal root ganglia. High traceruptake was also visible in the stellate ganglion, salivary glands andheart (FIG. 5A). In the shoulder joint, [¹⁸F]DCFPyL accumulation seemedto be restricted to the articular cartilage (FIG. 5A). The superiorcervical ganglion (SCG; volume approx. 9 mm³) was a simply recognizablestructure fitting in the 7 cm axial field of view of the Focus 220scanner together with the spinal ganglia, heart and the frontal part ofthe liver. It was sufficiently distant from bone structures. Thatallowed quantification of the SCG radioactivity accumulation even iftracer defluorination resulted in high ¹⁸F⁻ bone uptake. We thereforeused the SCG as a reference structure with a mean (±standard deviation)[¹⁸F]DCFPyL uptake of 20.2±5.8% ID (range 13.3-29.6% ID; n=6) and asignal-to-noise ratio of 6.7±2.6 (Table 1). The co-application of thePSMA-inhibitor 2-PMPA strongly decreased [¹⁸F]DCFPyL accumulation in allabove-mentioned PSMA-positive tissues (FIG. 5B, Table 1; decrease from20.2±5.8 to 4.6±1.8% ID in the SCG, n=3). At the same time traceraccumulation in the liver remained at the same level.

Uptake of 2-MeO-[¹⁸F]PSMA in SCG was higher than for [¹⁸F]DCFPyL(31.3±10.5 vs. 20.2±5.8% ID) (FIG. 5B). Together with the comparableunspecific uptake this resulted in a higher signal-to-noise ratio of8.2±1.7% ID. The bone uptake of the tracer was slightly lower incomparison to [¹⁸F]DCFPyL. Liver accumulation of 2-MeO-[¹⁸F]PSMA wasrather high. In contrast to the 2-methoxy substituted tracer,4-MeO-[¹⁸F]PSMA showed a low uptake and signal-to-noise ratio in the SCG(14.4±2.6% ID and 4.6±1.3, respectively) (FIG. 5C, Table 1).Interestingly, liver uptake of the tracer was significantly lower andthe bone uptake was higher as that of 2-MeO-[¹⁸F]PSMA (29.0±7.2 vs.119.3±8.3% ID and 19.2±2.1 vs. 9.9±1.9% ID, respectively).

While accumulation of [⁶⁸Ga]Ga-PSMA-HBED-CC in the SCG was significantlyhigher than that of [¹⁸F]DCFPyL (41.0±3.4% ID), high unspecific uptakein non-target target tissue resulted in a lower signal-to-noise ratio of4.5±0.1 (FIG. 5B). The image resolution was lower compared to that of[¹⁸F]DCFPyL, presumably, owing to the higher β⁺-energy of ⁶⁸Ga incomparison to ¹⁸F (1.9 vs. 0.6 MeV).

Next, [¹⁸F]AlF-PSMA-HBED-CC was studied (FIG. 5E). Surprisingly, despitethe previously described stability of the tracer, a high radioactivityuptake in bones (122.8±50.2% ID) indicated significant in vivodefluorination. The observed instability substantially limits theapplicability of [¹⁸F]AlF-PSMA-HBED-CC in clinical practice.

Among all tested PET tracers [¹⁸F]PSMA-1007 demonstrated the highestuptake in the SCG (FIG. 5E). A high unspecific uptake, however, led to asignal-to-noise ratio in the range comparable to [¹⁸F]DCFPyL (6.2±1.9vs. 6.7±2.6% ID for [¹⁸F]PSMA-1007 and

-   -   [¹⁸F]DCFPyL, respectively) (FIG. 5E, Table 1). Thus, bone uptake        of [¹⁸F]PSMA-1007 was higher and liver uptake was somewhat lower        as for [¹⁸F]DCFPyL (33.2±9.5 and 50.7±4.3% ID for [¹⁸F]PSMA-1007        and [¹⁸F]DCFPyL, respectively).

Example 12: PET Evaluation of [¹⁸F]1 and [¹⁸F]2 in Healthy Rats inComparison to Known PSMA Specific Tracers

Animals: Experiments were carried out in accordance with the EUdirective 2010/63/EU for animal experiments and the German AnimalWelfare Act (TierSchG, 2006), and were approved by regional authorities(LANUV NRW). Long Evans rats (250-590 g body weight) were used for thisstudy. Rats were housed in pairs in individually ventilated cages(NexGen EcoFlo, cages RAT1800 with 1805 cm² floor space and 41 cmheight; Allentown Inc., Allentown, N.J., USA) under controlled ambientconditions (22±1° C. and 55±5% relative humidity) on an inversed 12 hourlight/dark schedule (lights on 9:00 p.m.-9:00 a.m.). Food and water wereavailable at all times. Three rats received two, and one rat receivedthree different tracers. The other seven rats were measured with onetracer only. Each tracer was measured in three animals.

PET-imaging: Prior to PET measurements with PET probes, animals wereanesthetized (initial dosage: 5% isoflurane in 02/air (3:7), thenreduction to 2%), and a catheter for tracer injection was inserted intothe lateral tail vein. Rats were placed on an animal holder (Medres,Cologne, Germany), and fixed with a tooth bar in a respiratory mask.Dynamic PET scans in a list mode were performed using a Focus 220 microPET scanner (CTI-Siemens, Germany) with a resolution at a center offield of view of 1.4 mm. Data acquisition started with tracer injection(66±14 MBq in 0.5 mL i.v.), continued for 120 min and was followed by a10 min transmission scan using a ⁵⁷Co point source. For blocking studies2-(phosphonomethyl)pentanedioic acid (2-PMPA; 23 mg/kg) was addeddirectly to a radiotracer solution. Breathing rate was monitored andkept around 60/min by adjusting isoflurane concentration (1.5-2.5%).Body temperature was maintained at 37° C. by a feedback-controlledsystem. Following Fourier rebinning, data were reconstructed using aniterative OSEM3D/MAP procedure78 including attenuation and decaycorrection in two different ways: 1) 28 frames (2×1 min; 2×2 min, 6×4min, 18×5 min) for compilation of regional time activity curves; 2) 4frames (4×30 min) for visual display. Resulting voxel sizes were always0.38×0.38×0.79 mm.

Data analysis was performed using the software VINCI.79 Images wereGauss filtered (1 mm FWHM), and % ID was determined by dividing eachimage by the injected dose and multiplying the result by body weighttimes 100. Mean % ID values were extracted from each of the 28 framesand plotted over time.

First Clinical Experience with [¹⁸F]1

Owing to the favorable imaging properties of 2-MeO-[¹⁸F]PSMA in rats asmall pilot study with this tracer in 10 patients was conducted. Allpatients were examined with both [⁶⁸Ga]Ga-PSMA-HBED-CC and2-MeO-[¹⁸F]PSMA PET CT. In each case, both PET/CT scans were carried outwithin three weeks. Accordingly, six patients exhibited at least onePSMA-positive suspicious lesion detected by [⁶⁸Ga]Ga-PSMA-HBED-CC and/or2-MeO-[¹⁸F]PSMA PET/CT (FIG. 6 ). In four patients, at least oneadditional PSMA-positive lesion using 2-MeO-[¹⁸F]PSMA compared to thecorresponding [⁶⁸Ga]Ga-PSMA-HBED-CC image was identified (FIG. 6B). Inone patient inconspicuous in the [⁶⁸Ga]Ga-PSMA-HBED-CC scan, aPSMA-positive lesion was discovered by 2-MeO-[¹⁸F]PSMA PET/CT (FIG. 6C).

In the subsequent larger study, 124 patients with biochemical recurrence(BCR) of PCa were examined. In this patient cohort a sensitivity of83.0% was determined for 2-MeO-[¹⁸F]PSMA compared to 79.1% and 74.2%determined earlier for [¹⁸F]DCFPyL and [⁶⁸Ga]Ga-PSMA-HBED-CC,respectively.

Example 13: PSMA-PET Imaging of Patients with Biochemical Recurrence ofProstate Cancer

The study was conducted in accordance with the Institutional ReviewBoard. All patients gave written informed consent to PET imaging andinclusion of their data in a retrospective analysis. All procedures wereperformed in compliance with the regulations of the responsible localauthorities (District Administration of Cologne, Germany).

All measurements with [⁶⁸Ga]Ga-PSMA-HBED-CC and [¹⁸F]-2-MeO-PSMA werecarried out as described in the literature.^([14a, 15])

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BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Structures of PSMA-specific PET-Ligands used in this study.

FIG. 2 : Preparation of the novel PSMA specific probes, [¹⁸F]1 and[¹⁸F]2.

FIG. 3 : Synthesis of precursors 8, 9 and 16 for radiolabeling.

FIG. 4 : Cellular uptake of 2-MeO— and 4-MeO-[¹⁸F]PSMA (A and B,respectively), and [¹⁸F]DCFPyL in PSMA⁺ LNCaP C4-2 and PSMA⁻ prostatetumor cells. Data of cellular uptakes [¹⁸F]DCFPyL carried out inparallel with those of 2-MeO— and 4-MeO-[¹⁸F]PSMA are presented.

FIG. 5 : Uptake of different PSMA-specific tracers in healthy ratsmeasured by PET. Conditions: PET scanner (Focus 220, Siemens); 57-71 MBqtracer was injected. PET scans started 60 min after injection andcontinued for 60 min. A: [¹⁸F]DCFPyL; B: [¹⁸F]DCFPyL+2-PMPA (23 mg/kg);C: 2-MeO-[¹⁸F]PSMA; D: 4-MeO-[¹⁸F]PSMA, E: [⁶⁸Ga]Ga-PSMA-HBED-CC, F:[¹⁸F]AlF-PSMA-HBED-CC G: [¹⁸F]PSMA-1007 H: Shown sagittal section andlist of abbreviations.

FIG. 6 : FIG. 6 is provided in FIGS. 6 a and 6 b . Left column:[⁶⁸Ga]Ga-PSMA-HBED-CC PET-data, right column: 2-MeO-[¹⁸F]PMSA PET-datain the same patient. First row: Maximal intensity projections of thePET-data (darker black color reflects higher tracer uptake). Second row:Axial slices (caudal aspect) of the PET/CT fusion images (overlay ofPET-data on the CT data). PET-tracer uptake displayed in “hot metal”(brighter yellow color reflects higher tracer uptake). Bl: Bladder, Bo:Bowel, K: Kidney, L: Liver, Sa: Salivary glands, Sp: Spleen, Tu:Suspected tumor, U: Ureter, R: Right side, L: Left side.

FIG. 7 : Layout of 2-MeO-[¹⁸F]PSMA synthesis on FX—N-Pro synthesismodule.

FIG. 8 : Cassette for the production of [¹⁸F]1 on FASTab synthesismodule (GE).

FIG. 9 : Production of [¹⁸F]1 on FASTab synthesis module (GE).

The invention claimed is:
 1. A compound of formula (I):

wherein R is C₁-C₁₀ substituted or unsubstituted alkyl, or C₅-C₁₂unsubstituted or substituted aryl or heteroaryl.
 2. The compound offormula (I) according to claim 1 wherein R is C1-C4 substituted orunsubstituted alkyl.
 3. The compound of formula (I) according to claim 1wherein R is C1-C3 substituted or unsubstituted alkyl.
 4. The compoundof formula (I) according to claim 1 wherein R is methyl.
 5. A method forimaging of a PSMA-positive organ or tissue or both in a subject,comprising administering to said subject the compound of formula (I)according to claim 1 and obtaining and image of said organ or tissue orboth.
 6. The method according to claim 5, wherein said subject has apathological condition that is selected from the group consisting ofcancer, prostate cancer, re-endothelialization, neuropathic pain andatherosclerosis.
 7. A method for staging a pathological or physiologicalcondition associated with one or more PSMA-positive organs or tissues orboth of a subject, comprising administering to said subject the compoundof formula (I) according to claim 1 and staging said pathological orphysiological condition.
 8. The method according to claim 7, whereinsaid subject has a pathological condition that is selected from thegroup consisting of cancer, prostate cancer, reendothelialization,neuropathic pain and atherosclerosis.
 9. A method of making a compoundof formula (I) according to claim 1 from a compound of formula II andLys-C(O)-Glu comprising:

Y is Me₃N⁺Z⁻ or

Z⁻ is CF₃SO₃ or CF₃CO₂ R is C₁-C₁₀ substituted or unsubstituted alkyl,or C₅-C₁₂ unsubstituted or substituted aryl or heteroaryl, a) providingan aqueous solution of [¹⁸F]fluoride; b) loading of [¹⁸F]fluoride ontoan anion exchange resin; c) washing the anion exchange resin; d) dryingthe resin with flow of air or inert gas; e) elution of [¹⁸F]fluoridewith a solution of a compound of formula (II) in a polar aproticsolvent; f) if the elution was carried out by a C₂-C₆ alcohol as thesolvent, then diluting the reaction mixture with a polar aproticsolvent; g) heating the resulting solution at 30-70° C. for 1-30 minwhich furnishes a crude of a compound of formula [¹⁸F]III;

wherein R is as defined for formula II, h) purification of the compoundof formula [¹⁸F]III reversed phase solid phase extraction (RP SPE) asfollows: dilution of the above mixture with H₂O, loading the resultingsolution on a RP SPE cartridge, washing the cartridge with H₂O, elutionof the purified compound of formula [¹⁸F]III with a C₂-C₆ alcohol; i)elution of the purified compound of formula [¹⁸F]III directly to asolution of Lys-C(O)-Glu and a base in an anhydrous C₂-C₆ alcohol; j)heating the resulting solution at 30-70° C. for 1-30 min; k)purification of the resultant crude compound of formula I by RP SPE oralternatively RP HPLC; and l) optionally formulation.
 10. A method ofmaking a compound of formula (I) according to claim 1 from a compound offormula (IV) comprising:

wherein Y is Me₃N⁺Z⁻ or

Z⁻ is CF₃SO₃ or CF₃CO₂ R is C₁-C₁₀ substituted or unsubstituted alkyl,or C₅-C₁₂ unsubstituted or substituted aryl or heteroaryl, a) providingan aqueous solution of [¹⁸F]fluoride; b) loading of [¹⁸F]fluoride ontoan anion exchange resin; c) washing the anion exchange resin; d) dryingthe resin with flow of air or inert gas; e) eluting [¹⁸F]fluoride with asolution of a compound of formula (IV) in a C₂-C₆ alcohol; f)evaporation of volatiles; g) dissolution of the residue in a polaraprotic solvent; h) heating of the resulting solution at 40-130° C. for2-30 min; i) addition of 85% H₃PO₄ or 10 M HCl to a solution of aresultant crude compound of formula [¹⁸F]V;

wherein R is as defined for the compound of formula (IV), j) heating ofthe resulting mixture at 40-130° C. for 2-30 min; k) dilution of thereaction mixture and adjustment of the pH to 2.0-2.5 with an aqueoussolution of a base; l) RP HPLC purification by H₃PO₄ in aqueous EtOH asan eluent; m) dilution with isotonic saline, adjustment of the pH with abase; and n) sterile filtration.
 11. A kit or a cassette system forpreparing a compound of formula (I) according to claim 1, said kit or acassette system comprises (i) an anion exchange column; (ii) a reactionvessel; (iii) vials containing aliquots eluents; (iv) a vial containingan aliquot of a compound of formula II or IV

wherein Y is Me₃N⁺Z⁻ or

Z⁻ is CF₃SO₃ or CF₃CO₂ R is C₁-C₁₀ substituted or unsubstituted alkyl,or C5-C12 unsubstituted or substituted aryl or heteroaryl; (v) reagentvials wherein each reagent vial contains an aliquot of a reagent; (vi)optionally, one or more SPE columns for purification; (vii) optionally,a HPLC column for purification and, (viii) cleaning material for saidreaction vessel and said SPE columns.
 12. A pharmaceutical compositioncontaining at least one compound of formula (I) according to claim 1together with at least one pharmaceutically acceptable solvent,ingredient and/or diluent.
 13. A method for imaging prostate cancercells or prostate cancerous tissue, comprising administering to saidprostate cancer cells or prostate cancerous tissue the pharmaceuticalcomposition according to claim 12 and obtaining and image of saidprostate cancer cells or prostate cancerous tissue.
 14. A method ofmaking a compound of formula (I) according to claim 1 from a compound offormula II and Lys-C(O)-Glu comprising:

wherein Y is Me₃N⁺Z⁻ or

Z⁻ is CF₃SO₃ or CF₃CO₂ R is C₁-C₁₀ substituted or unsubstituted alkyl,or C₅-C₁₂ unsubstituted or substituted aryl or heteroaryl, a) providingan aqueous solution of [¹⁸F]fluoride; b) loading of [¹⁸F]fluoride ontoan anion exchange resin; c) washing the anion exchange resin; d) dryingthe resin with flow of air or inert gas; e) elution of [¹⁸F]fluoridewith a solution of a compound of formula (II) in a polar aproticsolvent; f) if the elution was carried out by a C₂-C₆ alcohol as solventdiluting the reaction mixture with a polar aprotic solvent; g) heatingof the resulting solution at 30-70° C. for 1-30 min which furnishes acrude of a compound of formula [¹⁸F]III;

wherein R is as defined for formula II, h) purification of the compoundof formula [¹⁸F]III by reversed phase solid phase extraction (RP SPE) asfollows: dilution of the resultant mixture with H₂O, loading theresulting solution on a RP SPE cartridge, washing the cartridge withH₂O, elution of the purified compound of formula [¹⁸F]III with C₂-C₆alcohol; i) elution of the compound of formula [¹⁸F]III directly to asolution of Lys-C(O)-Glu and a base in an anhydrous C₂-C₆ alcohol; j)heating the resulting solution at 30-70° C. for 1-30 min; k)purification of the resultant crude compound of formula I by RP SPE oralternatively RP HPLC; and and wherein the method does not comprise anevaporation step; and/or a deprotection step; and/or a neutralizationstep.
 15. A method of making a compound of formula (I) according toclaim 1 from a compound of formula (IV) comprising:

wherein Y is Me₃N⁺Z⁻ or

Z⁻ is CF₃SO₃ or CF₃CO₂ R is C₁-C₁₀ substituted or unsubstituted alkyl,or C₅-C₁₂ unsubstituted or substituted aryl or heteroaryl, a) providingan aqueous solution of [¹⁸F]fluoride; b) loading of [¹⁸F]fluoride ontoan anion exchange resin; c) washing the anion exchange resin; d) dryingthe resin with flow of air or inert gas; e) eluting of [¹⁸F]fluoridewith a solution of a compound of formula (IV) in a C₂-C₆ alcohol; g)dissolution of the residue in a polar aprotic solvent; h) heating of theresulting solution at 40-130° C. for 2-30 min; i) addition of 85% H₃PO₄or 10 M HCl to a solution of a resultant crude compound of formula[¹⁸F]V;

wherein R is as defined for the compound of formula (IV), j) heating ofthe resulting mixture at 40-130° C. for 2-30 min; k) dilution of thereaction mixture and adjustment of the pH to 2.0-2.5 with an aqueoussolution of a base; l) RP HPLC purification by H₃PO₄ in aqueous EtOH asan eluent; m) dilution with isotonic saline, adjustment of the pH with abase; and n) sterile filtration, and wherein the method does notcomprise an evaporation step; and/or a deprotection step; and/or aneutralization step.
 16. The compound of formula (I) according to claim1 wherein R is allyl, propargyl, phenyl or pyridyl.
 17. The methodaccording to claim 9 of making a compound of formula (I) from a compoundof formula II and Lys-C(O)-Glu comprising:

Y is Me₃N⁺Z⁻ or

Z⁻ is CF₃SO₃ or CF₃CO₂ R is methyl, ethyl, propyl, butyl, allyl,propargyl, phenyl or pyridyl, a) providing an aqueous solution of[¹⁸F]fluoride; b) loading of [¹⁸F]fluoride onto an anion exchange resin;c) washing the anion exchange resin; d) drying the resin with flow ofair or inert gas selected from the group consisting of He and Ar; e)elution of [¹⁸F]fluoride with a solution of a compound of formula (II)in a polar aprotic solvent selected from the group consisting of DMF,DMSO, MeCN and a C₂-C₆ alcohol or in a mixture thereof; f) If theelution was carried out by a C₂-C₆ alcohol as the solvent, then dilutingthe reaction mixture with a polar aprotic solvent selected from thegroup consisting of DMF, DMSO, MeCN, and aprotic solvent/C₂-C₆ alcoholmixture; g) heating the resulting solution at 40-50° C. for 2-7 minwhich furnishes a crude of a compound of formula [¹⁸F]III;

wherein R is as defined for formula II, h) purification of the compoundof formula [¹⁸F]III by reversed phase solid phase extraction (RP SPE) asfollows: dilution of the above mixture with H₂O, loading the resultingsolution on a RP SPE cartridge, washing the cartridge with H₂O, elutionof the purified compound of formula [¹⁸F]III with EtOH; i) elution ofthe purified compound of formula [¹⁸F]III directly to a solution ofLys-C(O)-Glu and a base selected from the group consisting of CsHCO₃,RbHCO₃, tetraalkylammonium phosphate, bicarbonate and carbonate in ananhydrous EtOH; j) heating the resulting solution at 40-50° C. for 2-7min; k) purification of the resultant crude compound of formula I by RPSPE or alternatively RP HPLC; and l) optionally formulation.
 18. Themethod according to claim 10 of making a compound of formula (I) from acompound of formula (IV) comprising:

wherein Y is Me₃N⁺Z⁻ or

Z⁻ is CF₃SO₃ or CF₃CO₂ R is methyl, ethyl, propyl, butyl, allyl,propargyl, phenyl or pyridyl, a) providing an aqueous solution of[¹⁸F]fluoride; b) loading of [¹⁸F]fluoride onto an anion exchange resin;c) washing the anion exchange resin; d) drying the resin with flow ofair or He or Ar; e) eluting [¹⁸F]fluoride with a solution of a compoundof formula (IV) in MeOH; f) evaporation of volatiles; g) dissolution ofthe residue in a polar aprotic solvent selected from the groupconsisting of DMF, DMSO, and MeCN; h) heating of the resulting solutionat 40-130° C. for 2-30 min; i) addition of 85% H₃PO₄ or 10 M HCl to asolution of a resultant crude compound of formula [¹⁸F]V;

wherein R is as defined for the compound of formula (IV), j) heating ofthe resulting mixture at 40-130° C. for 2-30 min; k) dilution of thereaction mixture and adjustment of the pH to 2.0-2.5 with an aqueoussolution of a base selected from the group consisting of NaHCO₃, Na₂CO₃,Et₃N, NaOH, Na₂HPO₄ and Na₃PO₄; l) RP HPLC purification by H₃PO₄ inaqueous EtOH as an eluent; m) dilution with isotonic saline, adjustmentof the pH with a base selected from the group consisting of NaHCO₃,Na₂CO₃, NaOH, Na₂HPO₄ and Na₃PO₄; and n) sterile filtration.
 19. The kitor a cassette system according to claim 11 for preparing a compound offormula (I), said kit or a cassette system comprises (i) an anionexchange column; (ii) a reaction vessel; (iii) vials containing aliquotseluents; (iv) a vial containing an aliquot of a compound of formula IIor IV

wherein Y is Me₃N⁺Z⁻ or

Z⁻ is CF₃SO₃ or CF₃CO₂ R is methyl, ethyl, propyl, butyl, allyl,propargyl, phenyl or pyridyl; (v) reagent vials wherein each reagentvial contains an aliquot of a reagent; (vi) optionally, one or more SPEcolumns for purification; (vii) optionally, a HPLC column forpurification and, (viii) cleaning material for said reaction vessel andsaid SPE columns.